The present invention relates to a method for the mutagenesis of eukaryotic nucleotide sequences, preferably from plants, algae and/or fungi, and to a method of generating such genetically modified eukaryotic cells.
A possible method of elucidating gene functions is the inactivation of genes and the subsequent observation of the effects on the metabolism or the phenotype of the modified system.
In particular gene inactivation by insertion mutagenesis has proved difficult in eukaryotic systems. The success of gene inactivation by homologous recombination in the host genome depends essentially on the ratio in which undesired, site-independent recombination occurs over site-specific homologous recombination. This ratio differs widely in eukaryotic organisms. A sufficiently high efficiency of the method for generating a large number of mutants is only described for lower eukaryotic organisms and starting from genomic DNA. Efficacies of above 10% have previously only been demonstrated in yeasts (Grimm and Kohli, 1988, MGG 215, 87-93; Struhl 1983, Nature 305, 391-397), various filamentous fungi (Fotheringham and Holloman, 1989, Mol. Cell Biol. 9, 4052-4055; Kronstad et al., 1989, Gene 79, 97-106; Paietta and Marzluff, 1985, Mol. Cell Biol. 5, 1554-1559; Timberlake and Marshall, 1989, Science 244, 1313-1317) and in Dictyostelium discoideum (De Lozanne and Spudich, 1987, Science 236, 1086-1091).
Homologous recombination in plants has previously been reported from Arabidopsis thaliana (Kempin et al., Nature 1998, 389, 802-803) for the AGL5 gene, an MADS box factor and for the TGA3 locus (Miao and Lam 1995, Plant Journal 7, 359-365). Here, however, only individual events were observed, and these permit no information on the statistic frequency (Puchta 1998, Trends Plant Sci. 3 (3), 77-80). Phenotypic changes were not observed in these cases. In the case of the AGL5 gene, one of 750 analyzed plants had a site-specific mutation (which corresponds to a recombination rate of 0.13%); in the case of the TGA3 locus, one event was found out of 2580 mutants (which corresponds to a recombination rate of 0.04%). It has been reported for Lotus japonica that no homologous recombination event was identified out of 18974 transformants (Thykjaer et al. 1997, Plant Mol. Biol. 35, 523-530).
Using genomic fragments of single-copy genes, knockout mutants were obtained in the moss Physcomitrella patens as a consequence of homologous recombination (Schaefer et al., 1997, Plant Journal 11 (6): 1195-1206). The homologous recombination rate was 90%. Again, no change in phenotype was observed in this case.
The utilization of genomic nucleotide sequences for generating mutants is disadvantageous inasfar as the DNA fragments may comprise a variety of operable units or linkages. Operable unit or linkage is understood as meaning the sequential arrangement of individual units and their linkage to each other, such as promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfill its intended function upon expression of the coding sequence. Site-unspecific insertion may have such a random modifying effect on, for example, promoters that a regulator with novel characteristics arises.
The analysis of the mutants may reveal further problems. If, for example, two genes are disrupted by a nucleic acid fragment, no definite conclusion regarding the involvement of a gene can subsequently be drawn since the contribution of an individual gene to the effect to be observed cannot be assessed. This would first have to be tested by more complicated analyses, such as, for example, sequencing of the genomic fragments used, and by determining the type of insertion in the plant and its effect on the genomic arrangement of modified gene segments.
The inactivation of genes by the insertion mutagenesis of genomic DNA by means of homologous recombination by known methods furthermore has a further considerable disadvantage. Thus, for example, the analysis of the entire genome of a host system requires the complicated construction of several thousand individual constructs of genomic origin, which constructs must also be retransformed individually into the host cell, before analysis of the mutants obtained by homologous recombination is made possible. Such genomic mutation approaches are thus not very economical and unsuitable for a routine throughput of experimental set-ups of substantial size, for example with the aim of obtaining a xe2x80x9csaturatedxe2x80x9d mutagenesis of the entire host genome.
It is an object of the present invention to provide a simple and efficient method for the mutagenesis in eukaryotic cells which no longer has the abovementioned disadvantages.
We have found that this object is achieved by a method for the mutagenesis of eukaryotic nucleotide sequences in which a genomic nucleotide sequence and/or cDNA sequence from eukaryotic organisms is transferred into a microorganism, the eukaryotic nucleotide sequence is genetically modified in the microorganism by sequence-independent insertion mutagenesis, and this genetically modified eukaryotic nucleotide sequence is subsequently isolated from the microorganism. Only one transposition event takes place per transferred eukaryotic nucleotide sequence. The transfer of eukaryotic cDNA is preferred.
In a further variant of the method according to the invention, the sequence-independent insertion mutagenesis is achieved by cointegrate formation during the conjugation between two microorganisms. Here, the eukaryotic nucleotide sequence is transferred into a microorganism which then acts as donor cell; the eukaryotic nucleotide sequence is subsequently mutated in the microorganism by sequence-independent insertion mutagenesis, during which process a cointegrate is first formed, the cointegrate formed is then transferred into another microorganism (receptor) via conjugation, the cointegrate is broken down in the receptor, during which process the eukaryotic nucleotide sequence is genetically modified by insertion of a heterologous nucleotide sequence, and this genetically modified eukaryotic nucleotide sequence is isolated from the microorganism. Heterologous DNA is to be understood in accordance with the invention as meaning DNA which does not naturally occur in eukaryotic organisms, i.e. which originates from prokaryotic organisms or, if appropriate, from viruses or phages, and which is replicated in microorganisms. In a preferred variant of the method according to the invention, cDNA sequences from eukaryotic organisms, preferably from plants, algae and/or fungi, are employed.
It is essential for the present method that the mutagenesis in the microorganism takes place with a high degree of efficiency, preferably with a relative frequency post-selection in a range of from approximately 90 to 100%, especially preferably of more than 90 to 99%, in particular 99.9%. In accordance with the invention, only one insertion event takes place per nucleotide sequence.
Since, in accordance with the invention, the mutagenesis of the eukaryotic nucleotide sequence takes place in a sequence-independent manner, that is to say randomly, by insertion of a heterologous nucleotide sequence, the method according to the invention is advantageously distinguished by the fact that the eukaryotic nucleotide sequence can be mutated without it being necessary to know its exact nucleotide sequence or specific restriction cleavage sites. Based on a selected region of a eukaryotic nucleotide sequence, a multiplicity of different (chimeric) eukaryotic nucleotide sequences, which merely differ from each other by the insertion site of the heterologous nucleotide sequence in the eukaryotic DNA, are generated in accordance with the invention by the sequence-independent mutagenesis.
Furthermore, the method according to the invention is also suitable for the mutagenesis of a very large number of different eukaryotic nucleotide sequences as are present for example in the form of a eukaryotic genetic library. A considerable advantage of the method according to the invention is that for example a complete genetic library can be genetically modified in a single mutagenesis set-up. In this context, a eukaryotic genetic library of genomic nucleotide sequences or else starting from cDNA may be used, the use of cDNA being preferred. In principle, both the use of natural eukaryotic nucleotide sequences and the use of chemically synthesized nucleotide sequences are possible. Chemical synthesis in this context can be effected starting from transcription products, as a rule total RNA or poly-(A)+ RNA, or else for example making use of protein sequences taking into consideration the codon usage of the eukaryotic cells.
In accordance with the invention, the eukaryotic genetic library is mutagenized homogeneously in a single mutagenesis set-up with a statistic probability in the range of from 90-100%, preferably 90-99%, especially preferably 99.9%.
Since the principle of the method according to the invention is based on sequence-independent mutagenesis, the origin of the eukaryotic DNA is of minor importance. The eukaryotic DNA employed may take the form of nucleotide sequences from plants, algae and/or fungi. In accordance with the invention, a nucleotide sequence from lower or higher plants is employed. A nucleotide sequence from lower plants of the genus Physcomitrella, Funaria, Ceratodon or Dicranum is preferably employed. A nucleotide sequence from Physcomitrella patens is especially preferably employed. The fungi can take the form of yeasts and/or filamentous fungi, preferably phytopathogenic fungi, especially preferably of the genus Fusarium. Other eukaryotic systems and DNA isolated therefrom are also feasible. The present invention is not limited by the above information on the origin of the DNA to be mutagenized.
In accordance with the invention, the eukaryotic nucleotide sequence is transferred into a suitable microorganism by methods known per se. To this end, the eukaryotic nucleotide sequence used can be introduced into a suitable vector, for example as shown in FIG. 1. A preferred vector is one which comprises natural nucleotide sequences isolated from eukaryotic cells and/or nucleotides synthesized chemically making use of eukaryotic DNA or in accorance with the codon usage of the eukaryotic cell. In further embodiments of the present invention, a vector comprising a eukaryotic nucleotide sequence and additional functional nucleotide sequences, for example as shown in FIG. 2, is transferred into a microorganism. Additional functional nucleotide sequences are understood as meaning in accordance with the invention for example a replication origin for multiplication in bacteria, a selection marker, recognition sites for restriction endonucleases suitable for cloning the eukaryotic DNA, and others. The vectors shown in FIG. 1, 2, 5 or 6 comprise examples of such constructs. The invention therefore also relates to vectors for use in a method of the abovementioned type with characteristics shown in FIG. 1, 2, 5 or 6. The vectors shown in FIG. 5 or 6 are derivatives of the vector shown in FIG. 1.
The present invention also relates to the genetically modified eukaryotic DNA resulting from the method according to the invention and/or to a population of resulting genetically modified DNA sequences. The invention furthermore relates to a vector comprising genetically modified eukaryotic DNA generated by the present method. A vector with characteristics are shown in FIG. 3 is preferred. In addition, the present invention also relates to a genetically modified microorganism comprising a genetically modified eukaryotic DNA of the above-described type or a vector comprising such a genetically modified eukaryotic DNA.
It is essential for the method according to the invention that the mutagenesis of the eukaryotic nucleotide sequence in a microorganism is effected by inserting a heterologous nucleotide sequence, preferably of prokaryotic origin. In accordance with the invention, the inserted prokaryotic nucleotide sequence especially preferably takes the form of a nucleotide sequence which has characteristics for transposition. In one embodiment of the present invention, a mini-transposon is used, which, in accordance with the invention, is reduced to the essential features required for transposition. In particular, it takes the form of what is known as the mino-transposon mini-Tn1000::nptll.
It is a further advantage of the method according to the invention that the prokaryotic nucleotide sequence used for insertion into the eukaryotic nucleotide sequence is only capable of transposition in prokaryotic organisms, but not in eukaryotic organisms. Accordingly, the mutations generated by the method according to the invention in the eukaryotic nucleotide sequence is distinguished by the fact that it is essentially stable.
Microorganisms which are employed in accordance with the invention in the above-described method are those which have the necessary characteristics for transposition and conjugation. The microorganism used in accordance with the invention comprises a transposase gene and/or a resolvase gene in replicable form. Preferably, the microorganism employed in accordance with the invention is distinguished by the fact that the transposase gene and/or the resolvase gene is under the control of an inducible promoter. Preferred in this context are, for example, IPTG-inducible promoters, arabinose-inducible promoters such as, for example, the araBAD promoter, or temperature-inducible promoters such as, for example, the phage xcex promoters PL or PR, which, in turn, are controlled by the thermosensitive repressor c1857. The genes required for transposition and conjugation may also be present in the microorganism in transient form, i.e. initially in cryptic form, and activated only under specific circumstances.
The characteristics regarding transposition and conjugation which have been illustrated above can be encoded chromosomally and/or conferred by vectors in the microorganism used in accordance with the invention. In accordance with the invention, the genetically modified microorganism comprises, in particular, a vector as shown in FIG. 4. This vector confers the necessary characteristics for conjugation between a donor cell and a receptor cell and for the transfer of nucleotide sequences. Moreover, this vector comprises a prokaryotic nucleotide sequence which is suitable for transposition. Preferably, the microorganism employed in the method according to the invention is a bacterium of the genus Enterobacteriaceae or Bacillaceae. The bacterium Escherichia coli is especially preferably used, in particular the non-pathogenic cell line E. coliK12.
The present invention furthermore relates to a method of generating genetically modified eukaryotic cells of plants, algae and/or fungi or their progeny, a genetically modified eukaryotic nucleotide sequence (which has previously been generated in accordance with the method according to the invention) being transferred into one of these abovementioned eukaryotic host cells, the transformed eukaryotic host cell is incubated under conditions which make possible and/or which trigger a targeted homologous recombination of the introduced genetically modified eukaryotic nucleotide sequence into the genome of the host cell, the eukaryotic host cells which comprise integrated into their genome a genetically modified eukaryotic nucleotide sequence of the abovementioned type are subsequently identified, and corresponding tissue of plants, algae and/or fungi and/or intact plants are regenerated from these cells.
The xe2x80x9cconditionsxe2x80x9d which make possible and/or which trigger a targeted homologous recombination in the eukaryotic cell which has been transformed in accordance with the invention are, for the purposes of the invention, for example the incubation of the cells in selection medium or a change in the culture temperature. In one embodiment of the invention, for example, a culture in kanamycin- and/or G418-containing culture medium and/or an increase in the culture temperature of from, for example, 37xc2x0 C. to 42xc2x0 C., can make possible and/or can trigger the insertion mutagenesis according to the invention of a eukaryotic DNA.
In addition, the homologous recombination in this method according to the invention takes place at a high degree of efficiency, which, however, depends on the length of the eukaryotic DNA fragment inserted. Preferably, the homologous recombination takes place with a relative frequency post-selection in a range of from approximately 0.1 to 99.9%, especially preferably of from 1 to 90%, in particular of more than 10%.
In accordance with the invention, the present method comprises the use of a genetically modified eukaryotic nucleotide sequence or a vector comprising a genetically modified eukaryotic nucleotide sequence which is prepared in the manner described further above, likewise in accordance with the invention.
As already described, the method according to the invention gives rise to a multiplicity of genetically modified, chimeric nucleotide sequences which differ from each other only by the insertion site of the heterologous nucleotide sequence. This multiplicity of genetically modified eukaryotic nucleotide sequences is exploited in accordance with the invention via homologous recombination for the generation of a multiplicity of genetically modified eukaryotic organisms, i.e. plants, algae and/or fungi. That is, the method according to the invention gives rise to a population of eukaryotic cells comprising differently genetically modified nucleotide sequences. In this context, the individual eukaryotic cells are distinguished by the fact that they are mutated within a narrow genetic region, but that they all differ from each other by the exact site of mutation. A further advantage of the method according to the invention is that it gives rise to a population of genetically modified eukaryotic cells whose genome is modified at different sites with a statistic probability in the range of from 90-100%, preferably more than 90-99%, especially preferably 99.9%, and which is thus suitable for establishing a eukaryotic mutant genetic library.
This method according to the invention is furthermore distinguished by the fact that the homologous recombination of the genetically modified eukaryotic nucleotide sequence into the genome of the eukaryotic host cell leads to a phenotypically discernible and/or measurably modified phenotype in the plant. That is, in a particular embodiment of the present invention, a eukaryotic nucleotide sequence can be chosen for use in the method according to the invention in such a way that the insertion mutagenesis is effected in particular within the coding region of an active gene. Owing to the insertion site, the functionality of the mutated gene loci can be modified to a different degree, which permits the detailed mapping of various domains of a gene. In accordance with the invention, the resulting genetically modified eukaryotic nucleotide sequence is destroyed specifically in one gene function, i.e. negative mutants are generated.
The integration site of the heterologous nucleotide sequence is identified in accordance with the invention by recombinant methods. Recombinant methods are understood as meaning, in the present context, all of the methods known per se by means of which the integration of known nucleotide sequences into the corresponding target sequence can be identified. Examples which may be mentioned are the hybridization with specific and suitably labeled probes, the identification of the target site via polymerase chain reaction (PCR), or the use of specific antibodies.
The method according to the invention is furthermore distinguished by the fact that the eukaryotic host cells employed are cells of lower and/or higher plants. Eukaryotic host cells which are preferably employed are cells of the genus Physcomitrella. Cells of Physcomitrella patens are especially preferably employed. Genetically modified algae and/or fungi are also generated in accordance with the invention by the method illustrated hereinabove. Genetically modified filamentous fungi are preferably generated, especially preferably phytopathogenic fungi, and in particular fungi of the genus Fusarium.
A further advantage of the method according to the invention is that the prokaryotic nucleotide sequence, or parts thereof, which is (are) used for insertion into the eukaryotic nucleotide sequence is (are) only capable of transposition in prokaryotic organisms, but not in eukaryotic organisms. Accordingly, the mutation, in the eukaryotic nucleotide sequence, which has been generated by the method according to the invention is distinguished by the fact that it is essentially stable.
The present invention furthermore relates to a genetically modified eukaryotic cell of plants, algae and/or fungi or its progeny generated by the abovementioned methods. Also encompassed are genetically modified eukaryotic cell tissue, reproductive material, seeds and/or spores of genetically modified eukaryotic cells of plants, algae and/or fungi. Also encompassed in accordance with the invention are intact plants and/or plant parts comprising genetically modified eukaryotic cells of the above-described type and/or capable of regeneration from such genetically modified cells. Preferred as genetically modified eukaryotic cell or its progeny is a cell of the genus Physcomitrella. This is especially preferably a cell of Physcomitrella patens.
The genetically modified eukaryotic cell according to the invention or its progeny is furthermore distinguished by the fact that it is modified in a phenotypically discernible form and/or has a positively measurably modified phenotype. In accordance with the invention, the genetically modified eukaryotic cell and its progeny bears the integrated heterologous (prokaryotic) nucleotide sequence predominantly in coding sequence regions. This is why the functionality of the genes concerned and of the gene products derived therefrom in the eukaryotic cells and their progeny which have been genetically modified in accordance with the invention is modified to a different degree as a function of the insertion site of the prokaryotic nucleotide sequence. In a particular embodiment of the present invention, the genes concerned, of the genetically modified eukaryotic cells and their progeny, no longer have any functionality. These mutations are also termed negative mutations or what is known as knock-out or loss-of-function mutations. The corresponding genetically modified eukaryotic cells and. their progeny are analogously termed negative mutants or knock-out mutants.
The genetically modified eukaryotic cells according to the invention are furthermore distinguished by the fact that they comprise genetically stably modified nucleotide sequences owing to sequence-independent (random) insertion of a heterologous nucleotide sequence. Preferably, the eukaryotic cell which has been genetically modified in accordance with the invention and its progeny comprise a stably inserted nucleotide sequence of prokaryotic origin. In this context, the genetically modified eukaryotic cells according to the invention and their progeny are distinguished by the fact that they comprise a nucleotide sequence which can be transposed into prokaryotic organisms and not into eukaryotic organisms, or parts of such a nucleotide sequence.
The present invention furthermore relates to the use of a vector as shown in FIG. 1, FIG. 5, FIG. 6 for cloning, and the subsequent mutagenesis of a eukaryotic nucleotide sequence. Also encompassed in accordance with the invention are the resulting vectors comprising a eukaryotic nucleotide sequence provided for mutation (hereinbelow termed derivatives of the vectors as shown in FIG. 5 or FIG. 6). The invention furthermore relates to the use of the vector as shown in FIG. 2 or derivatives of the vectors as shown in FIG. 5 or FIG. 6 for generating a genetically modified eukaryotic nucleotide sequence by above-described methods. The invention further relates to the use of the abovementioned vectors, preferably of the vectors as shown in FIG. 2, derivatives of FIG. 5 or FIG. 6 and a vector as shown in FIG. 4 for use in a microorganism for the mutagenesis of eukaryotic nucleotide sequences.
The genetically modified eukaryotic cells are furthermore employed for identifying functional nucleotide sequences in eukaryotic cells and/or for the characterization of eukaryotic nucleotide sequences of unknown function (functional genome analysis).
Moreover, the present invention relates to the use of genetically modified eukaryotic cells for use in fields of agriculture, pharmacy and/or medicine.